Orai1, the pore-forming subunit of the calcium (Ca2+) release-activated Ca2+ (CRAC) channel, plays a central role in store-operated Ca2+ entry (SOCE) in animal cells and thereby serves as a key regulator of intracellular Ca2+ homeostasis. Disruption of this tightly controlled process is associated with a wide spectrum of human diseases. Dysfunction can result from a multitude of remodeling mechanisms, including altered protein expression (up- or downregulation), assembly remodeling, or mutations. We focus in particular on Orai1 mutations, which have been linked to severe combined immunodeficiency (SCID) as a result of channel loss-of-function (LoF), as well as to disorders like tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) arising from gain-of-function (GoF) alterations. These mutation-induced functional defects can be attributed to a wide variety of disruptions in the complex activation cascade of the Orai1 channel. Under physiological conditions, Orai1 activation involves all four transmembrane (TM) domains and follows a sophisticated interaction mechanism that ensures accurate signal transmission from the protein periphery toward its central Ca2+-conducting pore. In this Review, we compile all currently known disease-associated Orai1 mutations, delineate the mechanisms by which they interfere with the activation cascade, and discuss their pathological relevance. Their widespread distribution across all the domains of this Ca2+ channel highlights that malfunctions at virtually any point along the Orai1 TM domain interfaces can profoundly impair its activation mechanism, ultimately leading to severe diseases.
Verma et al. (https://doi.org/10.1085/jgp.202513958) provide a mathematical model of the ocular surface that can be used to guide assessment and selection of treatment options for dry eye disease, favoring volume-directed over cell-directed therapies.
JGP study (Colmano et al. https://doi.org/10.1085/jgp.202513959) identifies passive leak currents as a likely common mechanism underlying hypomagnesemia with treatment-resistant seizures in patients with certain mutations in the Na+, K+-ATPase α1 subunit.
Early bioenergeticists who described the principles of chemiosmosis were aware that swelling of mitochondria was a likely and even frequent event, based on the large electrochemical gradient of K+ ions across the mitochondrial inner membrane. Swelling could be measured as a change in electron density by electron microscopy or by spectrophotometry in isolated mitochondria. The mitochondrial permeability transition (mPT) was originally described as an acute swelling change in mitochondria, later determined to be caused by the rapid opening of a pore (mPTP) defined biophysically and pharmacologically as a Ca2+- and voltage-dependent, cyclosporine A-sensitive large-conductance channel. The identity of the pore is controversial, but the ATP synthase c-subunit is a major candidate. In their breakthrough study (Akosah et al. https://doi.org/10.1085/jgp.202613979), they establish a novel dark-field imaging approach, allowing detection of mitochondrial swelling in living cells. Swollen mitochondria exhibit decreased light scattering and, therefore, microscopically "disappear." The cell-based imaging technique enables dissection of two separate processes, mitochondrial swelling, and depolarization. The authors demonstrate that K+ influx causes swelling but not immediate mitochondrial depolarization in wild-type cells, whereas in ATP synthase c-subunit knockout cells, Ca2+-dependent mitochondrial depolarization occurs without swelling, suggesting a lack of K+ influx. The results suggest that the ATP synthase c-subunit channel is the key member of a channel complex constituting the "swelling channel" of the mPTP.
TMEM63B, a mechanosensitive ion channel (MSC), is associated with severe neurodevelopmental disorders, including severe early-onset developmental and epileptic encephalopathy. Structure-function studies have shown that TMEM63B pathogenic variants, including V44M in transmembrane helix 0 (TM0) and T481N in TM4, cluster near the hydrophobic neck region of the ion permeation pathway, a region critical for gating and permeation. Notably, V44M and T481N convert TMEM63B into constitutive phospholipid scramblases without obvious effects on their MSC activity, revealing an unexpected channel-to-scramblase switch in these variants. To further define the mechanistic basis of this phenomenon, here we characterized I475del, a TM4 deletion variant near V44M and T481N. Unlike V44M and T481N, the I475del channel exhibited basal leak currents, enhanced mechanically activated currents, and elevated mechanosensitivity, altogether supporting its classification as a bona fide gain-of-function MSC variant. Like V44M and T481N, I475del also enabled constitutive phospholipid scramblase activity. However, unlike these variants, I475del uniquely displayed further potentiation of scramblase activity under hypotonic osmotic stress. In addition, mutating the key residues that control gating conformational changes abolished gain-of-function ion and lipid transport through I475del. Together, our results support a stratified molecular model of TMEM63B channelopathies in which the pathogenic variants progressively destabilize the hydrophobic gate, permitting lipid permeation at the resting state followed by force-induced ion and lipid cotransport. These findings advance the mechanistic understanding of TMEM63B function and TMEM63B-associated disease and provide a framework for developing therapeutic strategies targeting variant-specific pathologies.
Voltage-gated sodium (Nav) channel site-2 diterpenoid alkaloids, including potent toxin aconitine (ACO), are generally considered gating modifiers that stabilize activated/open conformations and impair fast inactivation. In contrast, the structurally related compound bulleyaconitine (BACO), which is used clinically in China, produces antagonist-like suppression of Na+ current. Compared with BACO, ACO has two additional hydroxyl groups and lacks a 4-methoxy substituent at aromatic ring. Atomic mechanisms underlying the opposite effects of BACO and ACO are unknown. In cryoEM structure 7w77, a BACO-related alkaloid (BACORA) binds in fenestration I/II of Nav1.3, exposing tertiary nitrogen toward the pore lumen. Here, BACO and ACO were docked into the channel. Monte Carlo energy minimizations (MCMs) yielded alkaloids' binding poses located akin, but not identical to that of BACORA. MCM profiles of a hydrated Na+ pulled through the pore in the presence of protonated alkaloids reveal electrostatic barriers that would block ion permeation. In contrast, in the presence of unprotonated ACO, hydrated Na+ was transiently chelated by nitrogen and two oxygen atoms and would pass by the toxin. In BACO, the absence of hydroxyl groups near nitrogen decreases the probability of Na+ chelation and would shift the equilibrium of H+/Na+ exchange at nitrogen toward the protonated state that would block ion conduction. These results suggest that stabilization of the open activation gate can be mechanistically decoupled from ion permeation when a ligand exposes protonated nitrogen to the pore lumen, creating a conduction barrier. The proposed mechanism of Na+ permeation through ACO-bound channel is analogous to that described for the Nav1.5 channel with batrachotoxin (BTX) in fenestration III/IV. Together, these findings explain the opposite effects of ACO and BACO on Nav channels and highlight analogous mechanisms of Nav activation by ACO and BTX despite their binding in opposite fenestrations.
KirBac1.1 is a prokaryotic homolog of mammalian inward-rectifier potassium (Kir) channels, and the functional equilibrium of KirBac1.1 is highly dependent on the type of membrane lipids. Like Kir channels, KirBac1.1 activity is also inhibited by cholesterol, a physiologically relevant lipid in human health and disease. Despite the slide helix is an important functional motif during lipid-dependent gating, the structural dynamics of the slide helix during cholesterol-induced channel inactivation is not well understood and is the focus of this work. Sequence analysis reveals that the slide helix of KirBac1.1 itself is a putative cholesterol-recognition motif. Liposomal K+ flux assays show that transport activity of the wild-type channel in PC/PG membranes is completely abolished at high concentration of cholesterol, whereas this is not observed in some of the single-cysteine mutants of the slide helix, indicating slide helix residues are critical for cholesterol sensitivity. Quenching of intrinsic Trp fluorescence upon increasing cholesterol concentration strongly suggests that the KirBac1.1 functional inhibition by cholesterol is possibly due to its direct interaction with the channel. Membrane penetration depth measurements using NBD-labeled slide helix residues clearly show relatively shallow membrane interfacial localization of the slide helix in cholesterol-containing membranes that is associated with significant structural dynamics changes and altered conformational heterogeneity. Based on distance measurements in varying membrane lipid compositions that stabilize the active and inactive conformations, we hypothesize that slide helix position in the membrane might possibly act as a "conformational switch" in regulating the KirBac1.1 function. These results involving dynamic lipid-protein interactions in lipid-dependent gating might be relevant for other Kir channels.
This modeling study explains the intracellular Ca2+ release that controls contraction of skeletal muscles of mammals as emerging from a statistical ensemble of couplons. Couplons are constituted by identical sarcoplasmic reticulum RyR1 channels, arranged in native "checkerboard" double-row pattern, with alternate channels (the V class) in physical contact with CaV1.1 tetrads resident in the electrically excitable T-tubular membrane. Activation starts from these and propagates allosterically to the RyRs devoid of CaV contacts (the C class). Both activations are imposed by changes in free energy of channel states, derived from electrical work on the CaV mobile charges. The allosteric connections, resulting in reciprocal energy changes, are interlaced, so that the couplon becomes a highly reactive continuum where allosteric effects may propagate from end to end. A robust inactivation of C channels maintains this highly excitable device under graded voltage control. The model reproduces within the variance of experimental observations the cell level records of Ca2+ flux, including voltage dependence and kinetics, under multiple combinations of voltage clamp pulses, and accounts for the conditioning effects known as "quantal release" and "deterministic inactivation." The simulations, which describe individual channel evolutions as stochastic Markov chains, are also consistent with the Ca2+ events recorded at the subcellular microdomain level as well as observations of coupled gating in bilayers. No explicit Ca2+ roles on activation or inactivation are required. The good model-vs.-experiment match enables inferences on mechanisms, their specializations in muscle tissues, and their variations in different taxa. It also encourages modular incorporation into more comprehensive models of cellular function.
Voltage-gated ion channels play important roles in many membrane-enclosed structures, including synaptic vesicles, endosomes, mitochondria, chloroplasts, viruses, and bacteria. Here, we study how compartment size and channel gating interact to shape voltage dynamics and ion content in sub-micron structures. In small compartments, assumptions underlying conductance-based (Hodgkin-Huxley type) models of membrane voltage must be relaxed: (1) stochastic gating of individual ion channels can quickly and substantially change membrane voltage; (2) these changes can equilibrate faster than channel state dwell times; and (3) ionic currents, even though as few as two channels, can substantially alter ionic concentrations. We adapted conductance-based models to incorporate these effects, and we then simulated voltage dynamics of small vesicles as a function of vesicle radius and channel density. We identified regimes in this parameter space with qualitatively distinct dynamics. We then performed stochastic simulations to explore the role of NaV1.5 in the maturation of macrophage endosomes. The stochastic model predicted dramatically different dynamics compared with a deterministic approach. Electrophysiology of nanoscale structures can be very different from larger structures, even when ion channel composition and density are preserved.
Various dominant disease phenotypes are caused by germline variants of ATP1A1, the gene for the ubiquitously expressed Na+,K+-ATPase (NKA) α1 subunit. What characteristics link specific variants to a particular disease phenotype remains unclear. We used heterologous expression in mammalian cells and Xenopus oocytes to evaluate the characteristics of α1 variants causing hypomagnesemia with treatment-resistant seizures: L302R, G303R, M859R, and W931R. Cell survival in the presence of micromolar ouabain was reduced in cells expressing ouabain-resistant versions of these variants, indicating defective function of the variants relative to WT. Fluorescently tagged WT-α1 expressed in HEK293 cells showed plasmalemma localization, while all variants displayed increased cytosolic signal. In variant-expressing oocytes under voltage clamp, each variant showed passive "leak" currents, which were unaffected by ouabain in L302R and M859R, partially inhibited by ouabain in G303R and enhanced by ouabain in W931R. Na+ and Cl‒ permeate through G303R and W931R. M859R and W931R presented small K+-activated ouabain-inhibitable NKA currents that were absent in L302R and G303R. Growth of cells in ouabain and enzymatic studies demonstrated Na+/K+-dependent active transport for W931R, albeit with reduced Na+ affinity. We solved the cryo-EM structure of W931R-α1β1 with bound ouabain at 3 Å resolution. The structure showed a distorted transmembrane segment M9, which causes a concavity in the detergent micelle, suggesting that similar distortions may provide a leak pathway at the protein-lipid interface. Our results demonstrate that besides a loss of NKA function, all four variants carry an aberrant leak current that probably underlies the seizures and the hypomagnesemia characterizing this phenotype.
Auxiliary β1 subunits play an important role in modulating voltage-gated Na+ (NaV) channel gating, thereby influencing their voltage-dependent behavior and overall function. Here, we examine β1 modulation of human NaV1.6 channels expressed in Xenopus laevis oocytes and identify a glutamate in the extracellular S3-S4 loop of domain IV (VSDIV; E1607) as a key determinant of β1-induced voltage shifts. Co-expression of β1 produces hyperpolarizing shifts in activation and steady-state inactivation (SSI) and accelerates macroscopic inactivation kinetics. Substitution of E1607 with lysine or alanine, but not the negatively charged aspartate, selectively attenuates β1-induced shifts in voltage dependence while preserving effects on slope factor, inactivation kinetics, and persistent current. Pharmacological perturbation of VSDIV with the α-scorpion toxin AaHII attenuates β1-dependent shifts in SSI without abolishing β1 effects on gating kinetics. Together, these findings identify an extracellular VSDIV element that contributes to β1-dependent control of voltage dependence and support a model in which β1 modulates NaV1.6 gating through VSDIV-linked transitions.
The volume-regulated anion channel (VRAC), formed by LRRC8 heteromers, regulates cell volume and is a promising therapeutic target. DCPIB is the most widely used VRAC inhibitor, yet its binding mechanism remains poorly defined. Although structural studies suggested a role of residue R103 in LRRC8A in DCPIB binding, its functional relevance has been unclear. Here, we combined electrophysiology in Xenopus oocytes and LRRC8-/- HEK293T cells with MD simulations and rational design of DCPIB analogs. Mutations of R103 abolished ATP-dependent block of VRAC but did not affect inhibition by DCPIB or the related inhibitor carbenoxolone (CBX), indicating distinct binding mechanisms. Simulations revealed two DCPIB-binding modes: one involving R103 and another engaging K51 and hydrophobic residues. In R103F mutants, DCPIB retained binding via π-stacking and alternative electrostatic contacts, explaining its persistent inhibition. Attempts to disrupt these interactions by mutating K51 led to nonfunctional channels. To further probe the role of electrostatic groups in the binding of DCPIB to VRAC, we synthesized derivatives varying charge and hydrophobicity. Functional assays and steered MD showed that analogs retaining the carboxylate group maintain high-affinity binding, whereas esterified forms exhibit rapid unbinding and reduced potency. Biphenyl substitutions increased binding energy but imposed steric penalties, suggesting a trade-off between affinity and accessibility. Our findings reveal an adaptable binding mechanism for DCPIB, contrasting with described lipid-displacement mechanisms for other VRAC inhibitors, and establish electrostatic complementarity as the primary determinant of high-affinity block. These insights provide a framework for rational design of next-generation VRAC inhibitors with improved specificity.
Variants in KCNC1 encoding the voltage-gated potassium (K+) channel subunit KV3.1 are an emerging cause of a spectrum of neurological disease including developmental delay/intellectual disability, ataxia, myoclonus, and epilepsy, including progressive myoclonus epilepsy and developmental and epileptic encephalopathy. Here, we report novel biophysical properties of a recurrent de novo missense variant in KCNC1 c.1196C>T (p.Thr399Met) associated with epilepsy, mild developmental delay, and nonprogressive ataxia. The variant is localized to the highly conserved pore region of the channel, and voltage-clamp electrophysiological recording demonstrated a complete loss of function, as seen in more severe forms of KCNC1-related disorders. When expressed with wild-type (WT) to mimic the heterozygous state of the variant as would occur in a disease context, current density was not markedly reduced; however, resulting currents displayed a ∼20 mV hyperpolarizing shift of the voltage dependence of activation along with slowed deactivation kinetics, consistent with gain of function. In order to better understand this “dominant-positive effect” exerted on the WT channel, we co-expressed the KV3.1 p.Thr399Met variant with the recurrent p.Ala421Val variant known to act via loss of function with near-complete absence of current and associated with severe KCNC1-related disease. Co-expression of the two nonfunctioning variants led to a mild rescue of K+ current compared with the KCNC1-p.Ala421Val variant alone, further supporting a dominant-positive effect. Both KCNC1-p.Thr399Met and p.Ala421Val displayed trafficking deficiency. These results suggest that inclusion of the p.Thr399Met variant in heterotetrameric KV3 channels alters the gating kinetics of WT channel subunits, and highlight the unique features of this variant. The apparent complete loss of function of the p.Thr399Met variant when expressed alone is inconsistent with the relatively mild clinical presentation of the patient, subsequently explained via the “dominant-positive” action when combined with WT subunits.
Odorants in the inhaled air bind to odorant receptors embedded in cilia of olfactory receptor neurons, triggering the opening of the two ciliary olfactory transduction channels; first, a cationic heterotetrameric cAMP-gated channel, followed by an excitatory Ca2+-activated Cl- channel TMEM16B (also called anoctamin 2), which gives rise to a transduction current. While cholesterol is known to modulate ion channels, its effect on the olfactory channels, specifically TMEM16B, is unclear. We addressed how membrane cholesterol regulates these transduction channels. We heterologously expressed the main subunit of the olfactory cyclic nucleotide-gated (CNG) channel, CNGA2, and the Cl- channel TMEM16B and recorded their function in control, cholesterol-depleted, and cholesterol-enriched membranes. Maximal cAMP- or Ca2+-evoked currents were not altered by varying cholesterol levels. TMEM16B showed a progressive reduction in current upon repeated exposure to Ca2+ ("rundown"), which was accelerated in cholesterol-depleted membranes but decelerated in cholesterol-enriched patches when compared with control patches. Maximal cAMP-gated currents were stable over time, but their sensitivity was altered, with lower and higher cholesterol levels increasing and decreasing the channel's sensitivity, respectively. Cholesterol depletion of patches excised from mouse olfactory cilia containing native channels entirely abolished the TMEM16B current, while the maximal current of the native CNG channel remained unaltered. Reduced cholesterol did not change the sensitivity of the native CNG channel. Thus, while both olfactory transduction channels are sensitive to changes in membrane cholesterol in differing ways, the precise channel stoichiometry and the specific membrane environment also contribute to their function.
Lysosomes are essential organelles in eukaryotic cells, required for autophagy, endocytosis, pathogen defense, cell signaling, and metabolic homeostasis. A model of lysosomal ion and water fluxes that captures the synchronized, interdependent operation of ion transporters and diffusion enables prediction of organellar responses to external perturbations and supports the design and interpretation of experiments. Particularly with the advent of organelle-targeted rhodopsin-based optogenetics, there is a pressing need to predict cellular outcomes following light-driven, specific ion transport in lysosomes and other organelles. Currently, no models of lysosomal ion balance fully align with existing experimental data or enable simulation of the organelle's response to stress. Here, we present an updated interactive model that recapitulates appropriate stress responses. We incorporated the functional activities of TPC1 and TMEM165, in addition to the previously included vATPase, ClC-7, TRPML1, and passive ion and water fluxes. The model remains robust during lysosomal maturation, membrane permeabilization, swelling, deacidification induced by vATPase inhibition or additional optogenetics-like proton efflux, and accumulation of weakly basic cationic amphiphilic drugs. Our simulations indicate that lysosomal Ca2+ depletion couples with organellar deacidification triggered by either increased proton leakage or vATPase inhibition, with potential involvement of TMEM165 weakening. Beyond predicting stress-response dynamics, the model enables investigation of highly selective perturbations that can be experimentally induced using optogenetics. Elucidating the mechanisms underlying stable, stress-resilient lysosomal function offers insights for developing anti-disease and antiaging interventions. Further model refinement critically depends on experimental characterization of the lysosomal NHE-like protein mediating sodium influx.
Scala et al. (https://doi.org/10.1085/jgp.202613986) use combined genetic and pharmacological approaches to demonstrate that Kir6.2/SUR2-containg KATP potassium ion channels are the functionally relevant isoform that modulate contractile behavior of fast-twitch muscle during fatigue.
JGP study (Lukyanenko et al. https://doi.org/10.1085/jgp.202513844) shows that, in the absence of full-length dysferlin, its C2A domain alone can support normal Ca2+ signaling and sarcolemma repair, suggesting that it could be used in gene replacement therapies.
Vesicular monoamine transporters (VMAT) are proteins essential for life. They play central roles in neurotransmission and hormone storage and are targets of neuroactive drugs. This retrospective chronicles the decades-long journey of studying VMAT, from early biochemical explorations to the recent surge of structural information. We delve into the serendipitous discoveries, methodological innovations, and collaborations that enhanced our understanding of monoamine storage and synaptic transmission. Importantly, this narrative highlights that a degree of initial "optimistic ignorance"-a failure to fully appreciate the system's complexity-can, paradoxically, be a driving force in scientific exploration. Our early, naïve assumption about VMAT's abundance, while ultimately an oversimplification, was instrumental in launching the project to identify, purify, and characterize the protein. An unprecedented flood of cryo-EM structures now provides invaluable molecular insights into the molecule's intricate workings, synergistically complementing functional studies and offering the crucial framework needed to mechanistically link structure to its function.
A recent publication by our research team has introduced a novel group of hHv1 inhibitors, the 5-phenyl-2-aminoimidazoles. To guide rational drug design of selective derivatives, our goal was to map the molecular mechanism of compound 13 (i.e., NZ-58), a potent member of the series. For this, we utilized site-directed mutagenesis and conventional patch-clamp technique. Applying NZ-58 at various membrane potentials, and using different pulse frequencies, we could unveil the binding mode, which showed closed-state inhibition and no use dependence. The changes in inhibition of mutated hHv1 constructs confirmed the binding pocket suggested by molecular docking comprising countercharges D112 and D185. Combining the results of ionic and gating current measurements revealed an interesting molecular mechanism that traps the S4 helix. Knowing the interacting partners of NZ-58, this mechanism aligned well with previously found functional models of hHv1. By integrating pharmacological and biophysical perspectives, this work lays the groundwork for future advancements in rational drug design.
